Human bone obtained from cadaveric donors is typically procured under sterile conditions in an operating suite environment of local hospitals. The bone is stored frozen until it is further processed into small grafts under similar sterile conditions, or under clean-room conditions.
Procurement and processing of human tissues is typically performed by groups certified by the American Association of Tissue Banks under standard operating procedures for the processing of each specific bone graft. Large bones such as the femur are thawed and debrided of excess tissue prior to being cut into smaller grafts.
Prior art methods of processing smaller grafts include cleaning of bone marrow from the cancellous bone spaces using mechanical means as disclosed in U.S. Pat. No. 5,333,626, including soaking, agitation, and/or lavage with pulsatile water flow under pressure. Cleaning may involve reduced or elevated temperatures, for example 4.degree. C. to 65.degree. C., and may also include the use of detergents or decontaminating agents. Typically, pulsatile water flow under pressure, even with the use of detergents or alcohols, only permits removal of that bone marrow which is accessible by the pulsatile flow of water, i.e. the immediate surface part of the cut bone graft.
U.S. Pat. No. 5,513,662 discloses cleaning large bone grafts, for example, a proximal femur, by subjecting the femur itself to a negative pressure gas atmosphere. 662' claims that the graft is cleaned due to leakage of the bone marrow elements and lipids, such leakage induced by placing the cut bone graft with or without holes drilled into the graft, in a bell jar and drawing a vacuum.
Although current donor screening protocols have been suggested to reduce the potential for transmission of the HIV virus through allograft tissues to less than one chance in from 1 to 10 million, the post procurement processing of allograft tissues is currently a manual process involving individual technician handling of the tissues. Since bone marrow represents the largest potential reservoir for any HIV virus present in an allograft bone tissue, it is expected that the process step of removal of bone marrow from the allograft bone tissues poses the greatest risk of transmission to the technician doing the processing. For example, a human femur may provide individual bone grafts consisting of portions of the bone shaft, femur head, proximal and/or distal femur grafts, and dividing the femur into these parts prior to removal of bone marrow increases the potential for process associated scattering of bone marrow elements within the processing room.
Bone marrow elements include hematopoietic progenitor cells, i.e., those stem cells that will ultimately differentiate into red blood cells, white blood cells, and platelets, among others. These stem cells are rich in major histocompatibility antigens (i.e., MHC antigens) that function in immune responses. Current processing techniques do not effectively remove bone marrow from the less solvent-accessible cancellous bone spaces within bone grafts, such as, for example, the trochanter portion or the femoral head area of the proximal femur, because current processing techniques rely upon soaking procedures which may or may not include agitation.
Typically, hydrogen peroxide is used to oxidize the colored elements within the bone marrow, which results in a cleaner appearance. However, such bone often still contains bone marrow which is extremely inmunogenic.
Further, most bone grafts are currently stored in the freeze-dried state. Freeze-drying removes water from the grafts, but lipid elements present in the membranes of the bone marrow cells and in vesicles present in adipocytes (i.e., fat storage cells) typically leak from the grafts after being placed in their final storage and distribution containers. Thus, these residues often give the appearance that the graft itself is not clean.
Cleaning of bone marrow from small bone grafts has been described in the scientific literature and in brochures and documents made public by groups involved in the procurement and processing of human tissues. A for-profit public corporation, Cryolife, Inc. (Marietta, Ga.) promotes a bone cleaning process designated as VIP.TM. (Viral Inactivation Process) and claims that the process provides "Cleaner bone through mechanical removal of debris and tissue such as bone marrow, lipids and blood components" and "Safer bone through inactivation of pathogens such as HBV and HIV (greater than 5-log kill) as well as bacteria and fungi" (Cryolife Orthopedics, Inc. brochure Feb. 12, 1992; Cryolife literature directed to Organ and Tissue Procurement Program Directors dated Feb. 20, 1992).
A second, for-profit publicly held corporation, Osteotech, Inc., Shrewsbury, N.J., describes a bone graft cleaning process called Permein.TM. ("a combination of ethanol and non-ionic detergent"; Mellonig, J. T., Prewett, A. B., and Moyer, M. P. J. Periodontol. December 1992, vol. 63, pp 979-983). This process involves the use of a solution of ethanol and detergent to clean bone grafts. Details of the process and detergents utilized are not currently available. Bone is soaked in the solution and it is claimed that the combination of ethanol and detergent facilitates permeation of the solution into bone. The process has been demonstrated to clean small cut-bone grafts and to be capable of inactivating the HIV in bone allograft (finely ground bone) (Mellonig, Prewett, and Moyer, J. Periodontology, December, 1992, 63: 979-983).
U.S. Pat. No. 5,556,379 describes a process for removing substantially all bone marrow from a large essentially intact bone using a vacuum mediated flow of solvent into the bone via the natural foramen and cartilaginous ends. This patent is directed primarily at the cleaning of bone marrow from large bones prior to their being cut into small cut bone grafts. U.S. Continuation-in-Part patent application Ser. No. 08/620,856 describes a composition for cleaning bone, U.S. Continuation-in-Part application Ser. No. 08/619,412 describes a process for cleaning large essentially intact bone grafts whereby a combination of positive and negative pressure is used to remove bone marrow from essentially intact bones prior to their being processed into cut bone grafts, U.S. Continuation-in-Part application Ser. No. 08/646,519 describes the use of ultrasonic cavitation in the cleaning of large essentially intact bone prior to their being processed into small cut bone grafts and for cleaning of small cut bone grafts, and U.S. Continuation-in-Part application Ser. No. 08/646,520 describes a recirculation method of cleaning large essentially intact bone prior to their being processed into smaller cut bone grafts. All of these patents and continuation in part patent applications are hereby incorporated in their entirety in this patent application.
The presently claimed process differs from prior art processes, including those disclosed in U.S. Pat. Nos. 5,333,626 and 5,513,662, in that the present process uses centrifugal force to remove bone marrow elements from the bone graft. The bone marrow elements are impelled outward from the bone graft from a center of rotation. Neither a "high pressure washing condition" as disclosed in U.S. Pat. No. 5,333,626 nor exposure of the graft to a negative pressure gas atmosphere, is used.